Collagen peptide supplementation before bedtime reduces sleep fragmentation and improves cognitive function in physically active males with sleep complaints

Our sample size was determined from an a priori power analysis (G*power 3.1.9.2, Microsoft Windows [44]) for our two primary outcome measures, SOL, and sleep efficiency (SE), as measured by polysomnography (PSG). Based on previous research [10, 19, 45], we used a mean difference of 10 min (10 min SD) for SOL, and 4% for SE (4% SD) to estimate our sample size. With two tails, and an alpha of 0.05, it was calculated that we needed ≥ 10 participants to achieve 80% statistical power.

Thirteen athletic males with sleep complaints provided written informed consent to participate in this study; characteristics are presented in Table 1. Height and body mass were recorded at familiarisation. Inclusion criteria; 1) males 18–35 years; 2) Athens Insomnia Scale (AIS) score ≥ 6, indicating difficulty sleeping; 2) morning-eveningness questionnaire (MEQ) score of 31–69, indicating neither extreme morning or evening person; 3) scheduled exercise ≥ 3 occasions per week in the past 6 months. Participants completed a health history questionnaire and were excluded if they used sleep medications, had a diagnosed sleep disorder, reported excessive use of substances that negatively affect sleep such as drugs, alcohol, or smoking, regularly used anti-inflammatory medications (within 2 weeks of participation), had a food allergy, or any other contraindication to the study procedures. The protocol received ethical approval from Loughborough University School of Sport, Exercise, and Health Sciences Ethics Committee (ethics number: 1781) and was conducted in accordance with the Declaration of Helsinki. The study was pre-registered on the Open Science Framework: osf.io/vjf8y.

Prior to the supplementation trials, participants completed a familiarisation trial, in which they had their physical activity and sleep monitored for 7 days and nights in their home. This was to characterise exercise and sleep schedules (i.e., bedtimes and wake times) for the supplementation trials, and to familiarise them with the protocol and equipment. No supplements were consumed for these 7 days. Sleep was monitored via actigraphy and subjective sleep quality for 7 days, and then PSG on the final 2 nights (nights 6 and 7).

Following this familiarisation trial, participants were randomised, in a double-blind manner, to consume a 200 ml bolus of a CP based or a placebo control (CON) drink 1 h before bedtime for 7 days (trials were separated by ≥ 7 days). The trial order was randomly generated using online software (Graphpad Prism, CA, US). We provided CP for 7 days as this duration of intake has previously been shown to positively influence physiological markers [42, 46].

Sleep monitoring during both trials was the same as the familiarisation trial. Surveys to assess fatigue and sleepiness were also completed daily. On day 7, participants reported to the lab in the evening (≥ 16:00), had a venous blood and urine sample collected, consumed a telemetric temperature pill (E-celsius Performance Pill, Body Cap, France) and performed a battery of cognitive function tests. Although the timing of pill ingestion (1 – 12 h before) does not appear to interfere with the validity of the sensor [47], consistent with previous studies, we provided the pill 5–8 h before our first recording [46, 48], to ensure passage from the stomach. These telemetric pills have been shown to have excellent reliability and validity [49] and are considered the industry standard for temperature monitoring [50]. Participants returned to the lab after an overnight fast on the morning of day 8 (08:00–10:00) to have another blood and urine sample collected and to repeat the cognitive function tests. Participants were instructed to keep their bedtime and wake times the same as in their familiarisation week. On day 7 of both trials, participants followed a low protein diet and consumed a standardised evening meal. No exercise was permitted on day 7; participants were instructed to replicate their dietary intake and physical activity patterns recorded during the familiarisation week.

Participants consumed 15 g/day of CP derived from bovine hide or an inert, taste matched, CON as a 200 ml liquid bolus, without food, ~ 1 h before their scheduled bedtime (as recorded in the familiarisation trial) for 7 days. A 15 g CP dose was selected as it contains ~ 3.5 g of glycine, an amount shown to enhance sleep quality [19, 51]. The dose was consumed 1 h pre-bed, as (1) a previous study with glycine showed this improved polysomnographic sleep [19] and, (2) plasma glycine concentrations peak 30–60 min post CP intake [52]. Both supplements were provided in identical opaque bottles by Rousselot BV (Ghent, Belgium). The investigators remained blinded until data analysis was completed. No participants reported adverse effects from the supplements.

The AIS is an eight-item survey that assesses sleep difficulty in the past month; a score of ≥ 6 is indicative of poor sleep [53]. The AIS is based on diagnostic criteria from the International Classification of Diseases Tenth Revision (ICD-10) and has excellent reliability and validity in adult populations [53, 54]. It also has excellent reliability in athletic populations [55]. The MEQ measures chronotype, differentiating individuals into morning and evening types [56]. A score of < 31 indicates an extreme evening type; a score of > 69 indicates an extreme morning type. We excluded either extreme morning or evening types to increase the homogeneity of our sample.

Participants used sleep diaries to self-report their time in bed, bedtime, SOL, number of awakenings, length of awakenings, wake up time, and get up time during the trials. Any day-time naps were also recorded. Training diaries were used to record the intensity and volume of scheduled exercise completed each day. Intensity (1 = very low intensity, 10 = maximum intensity) was multiplied by duration (min) to give a composite score of training load [57]. Participants also wore wristwatch actigraphs (ActiGraph GT9X Link, Actigraph, FL, US) on their non-dominant wrist to measure physical activity levels and sleep. The participants were instructed not to remove the watch apart from to shower or bathe; if the watch was removed at any other point, they were instructed to record this in their sleep diaries. Actigraphy data was sampled at 60 s epochs and analysed using the Cole Kripke algorithm [58] on ActiLife software (ActiGraph, FL, US). Bedtime and wake up times from the sleep diaries were used for data analysis. Sleep variables analysed from actigraphy were time in bed (TIB), total sleep time (TST), SOL, SE, wake after sleep onset (WASO) and average wake length. Physical activity was analysed as total time (min) spent in moderate to vigorous activity using the cut off points suggested by Freedson and colleagues [59].

Polysomnographic sleep was measured on nights 6 and 7 of each trial with an ambulatory monitor (Embletta MPR with ST + Proxy, Natus Medical, CA, US). Night 6 served as a familiarisation collection only and was not used for analysis. Six electroencephalogram (EEG) channels (F3-M2; F4-M1; C3-M2; C4-M1; O1-M2 and O2-M1), two electrooculogram (EOG) channels (LEOG-M2 and REOG-M1), a submental chin electromyogram (EMG) (3 EMG electrodes) and the PSG unit were fitted in the lab ~ 6–8 h before sleep. All EEG sites were located using the international 10–20 system. The PSG was scheduled to start recording 30 min prior to bedtime. All recordings were collected at home; participants were instructed to sleep in the same bed, in the same environmental conditions and expose themselves to the same mental stimulation (e.g., phone use, television) in the 1 h before bedtime on each trial. Any deviances were reported in their subjective sleep diaries. Home-based PSG is considered as valid and reliable as clinic-based PSG set ups [60, 61]. Recordings were subsequently exported in EDF format and downloaded into Domino software v3.0.0.3 (SOMNOmedics, Germany) where they were manually scored in 30 s epochs using the standard adult criteria from the American Academy of Sleep Medicine (AASM) [62] by registered sleep physiologists with ≥ 20 years’ experience. Each recording was labelled with a different non-identifiable code so that the scorers (RNK and KMA) were blinded to participant identity and trial. As with actigraphy, lights out was marked as the time participants first attempted to go to sleep and lights on was marked as the time participants woke up for the final time (both from sleep diaries). To assess intra-rater reliability, 3 blinded PSG recordings were scored twice by RNK. To assess interrater reliability, KMA independently scored 3 PSGs previously scored by RNK. Each recording was scored for all outcomes presented in Table 2.

Subjective sleep quality was recorded with a 7-point Likert scale, where 1 = extremely poor and 7 = extremely good [23]. Sleepiness was measured with the Karolinska Sleepiness Scale (KSS), where 1 = extremely alert, and 10 = extremely sleepy, can’t stay awake [63]. Fatigue was measured with a 100 mm visual analogue scale, where 0 mm = not fatigued at all and 100 mm = extremely fatigued [64]. Sleep quality was recorded upon waking, while KSS and fatigue scores were recorded upon waking, at 14:00 and 1 h before bedtime. These were recorded daily with 7-day averages used for analysis.

Participants completed a series of cognitive tests that took approximately 10 min on a laptop. All tests were preceded by 3–5 practice trials. The first test was simple reaction time (RT), where participants were instructed to press the spacebar when a green circle appeared on a blank screen. The second test was choice RT, where participants were presented with right or left arrows and were required to press the arrow key that matched the direction on the screen. Average and fastest reactions times of correct responses (ms) were analysed for both; there were 10 and 14 attempts for simple and choice RT, respectively. Participants then completed the Digit Span Test, which measures attention and short-term recall [65]. To complete this test, a sequence of 3–14 digits were flashed on the screen and then participants were instructed to type the digits in the correct order. The length of the longest correct span over 12 trials was used for analysis. Finally, participants completed the Stroop Color Word Test, which measures attention and processing speed, but primarily the ability to inhibit cognitive interference [66, 67]. For the baseline level, participants were presented with names of colours (written in black) on a white background and instructed to correctly identify the name of the colour by pressing the right or left arrow. For the interference level, participants were presented with colored names on a white background and were required to correctly identify the font color, ignoring the incongruent colour named on the screen (e.g., if “green” is written in red font, then red is the correct answer). Participants had to respond as quickly as possible using left or right arrow keys. Outcomes were the proportion of correct responses, and average RT of correct responses (ms), for the baseline (14 trials) and interference (20 trials) levels.

Participants recorded their dietary intake on day 7 of the familiarisation trial and were instructed to replicate this intake on the two supplementation trials. On this day they were instructed to limit their intake of protein-rich foods (a list was provided), avoid consuming any caffeine after 11:00, and attend the lab ≥ 2 h post-prandial. Participants were given a low-protein evening meal to consume at home consisting of 1 × 250 g pack of White Golden Vegetable Rice (Tesco, PLC, Herts, UK) and 2 × 37 g Nutrigrain Blueberry bars (Kelloggs, Michigan, US). After this meal they avoided any food or fluids other than water and their respective supplements 1 h before bedtime until after their lab visit on day 8.

Samples were analysed for high sensitivity c-reactive protein (hs-CRP), interleukin-6 (IL-6), cortisol, and urine samples for normetanephrine, as these are sensitive to changes in sleep quality [68]. IL-6 was analysed in plasma using a commercially available ELISA kit (R and D Systems, MN, US); the inter-assay coefficient of variation (CV) was < 6.9%.

Serum cortisol and urine normetanephrine were measured using Liquid chromatography tandem mass spectrometry (LC–MS/MS) methods by a Waters Acquity I-class UPLC system coupled to the Xevo TQ-XS tandem mass spectrometer (Waters Corp., Milford, MA, USA) operated in positive electrospray mode. Serum cortisol was extracted using the Extrahera™ automation system (Biotage, Uppsala, Sweden) under positive pressure supplied by a nitrogen generator. In a 96-position 2 mL deep well plate, 200 μL of calibration standards (Chromsystems, München, Germany NIST SRM971 traceable), quality control materials, and serum samples were added to each well with 50 μL internal standard solution containing cortisol-d4 (IsoSciences, King of Prussia, PA, USA). 200 μL of isopropanol:water 50:50 (v/v) was then added to dissociate the binding proteins. After mixing, the samples were loaded onto ISOLUTE® supported liquid extraction (SLE +) 400 μL plate (Biotage). Elution was carried out by adding two cycles of 750 µL of methyl tertiary butyl ether (MTBE). The eluents were collected into a corresponding deep well plate. Positive pressure was applied at each stage to remove residual solvent. Samples were then dried to completeness under a gentle stream of nitrogen gas heated to 60 °C, then reconstituted with 100 μL of 50:50 methanol/water before being vortexed and sealed. 10 μL of the extract was injected into the LC–MS/MS. Chromatographic separation was achieved using a CORTECS™ core–shell C18 50 × 2.1 mm, 2.7 µm, reversed-phase (Waters Corp., Milford, MA, USA) column heated at 30˚C. Mobile phases used were (A) water and (B) methanol in 0.1% formic acid, pumped at the flow rate of 0.4 mL/min in 70:30% (A:B), gradually increased to 100% (B) then returned to the starting gradient at 4 min. Tandem mass spectrometry detection was based on the mass-to-charge (m/z) precursor to product ion transitions specific to each compound: cortisol (363.3 > 121.2) and cortisol-d4 (367.3 > 121.2) as an internal standard. The inter-assay CV for serum cortisol was < 6.0% across the assay range of 0.3–806 nmol/L.

Urine normetanephrine was extracted using solid phase extraction method (Chromsystems Biogenic amino #80,600, München, Germany) as per the manufacturer’s instruction. The assay was calibrated using human urine-based calibration standards and controls (Chromsystems) that are traceable to certified reference materials. m/z transitions were: normetanephrine (166.1 > 106.1) and normetanephrine-d3 (169.1 > 109.1) as internal standard. The inter-assay CV for normetanephrine was 3.4–6.2% across the assay range of 20.5–10,311 nmol/L. Urine results obtained from LC–MS/MS analysis were adjusted for variations in renal function by dividing by urine creatinine. Urine creatinine was analysed using Roche 2nd generation kinetic colorimetric assay based on the Jaffé method performed on the COBAS® C501 analyser (Roche, Burgess Hill, UK). The inter-assay CV was < 2.1%. The final results are expressed as nmol/L per mmol/L creatinine.

Serum hs-CRP was measured using a particle-enhanced immunoturbidimetric assay analysed on the COBAS® C501 analyser (Roche, Burgess Hill, UK). The inter-assay CV was < 2.6% across the assay working range 1.43–190 nmol/L.

Data analysis was performed with SPSS (IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp). Normality was assessed by visually inspecting histograms and skewness and kurtosis and homogeneity of variance for linear mixed models by plotting the residuals against the predicted values. Variables collected via PSG, actigraphy, and subjective sleep diaries as well as physical activity indices were measured with paired t-tests; any variables not normally distributed were analysed with non-parametric Wilcoxon signed rank tests. Actigraphy and subjective sleep data is an average of the 7 supplementation nights. Core temperature, KSS, fatigue, and all cognitive function and blood outcomes were analysed with linear mixed models. Mixed models were preferred to repeated measures ANOVAs because they better account for missing data. Time, condition (CP vs. CON), and time*condition interaction effects were included as fixed factors, and participant as a random factor. Models were run using the keep it maximal approach recommended by Barr et al. [69]; therefore, random intercepts and random slopes were initially included for all within-subject variables. If convergence wasn’t achieved, then the random effect’s structure was simplified. The covariance type was scaled identity and the model estimation was restricted maximum likelihood. Post hoc tests were adjusted for multiple tests using the Bonferroni correction. Intra and inter-rater reliability for PSG variables were assessed with intraclass correlation coefficients (ICC), using absolute agreement and a two way mixed-effects model (as in SPSS); values < 0.50 were considered to have poor reliability, 0.50–0.75 moderate reliability, 0.76–0.90 good reliability, and > 0.90 excellent reliability [70]. P < 0.05 was considered statistically significant. Hedges g effect sizes are reported for normally distributed data; rank biserial correlations (r_rb) are reported for non-normally distributed data. 95% confidence intervals (CI) are presented for the difference in means (CON - CP in all tables) or, if non-parametric tests were performed, the median of the differences, using the Hodges-Lehman estimator.

For intrarater ICC values, apart from N3 (%) which showed moderate reliability (0.61–0.64), all other variables showed good reliability (≥ 0.83). For interrater ICC values, all variables had good reliability (≥ 0.86), apart from N2 (%), which showed moderate reliability (0.58).

Table 2 displays the data collected via PSG. We failed to get traces for 1 participant and therefore this data is for 12 participants. The data presented is from night 7 of the 7-day supplementation period. During the CON and CP trials participants went to bed at 22:57:13 ± 01:07:14 and 22:50:19 ± 01:09:11, respectively; they woke at 07:10:36 ± 00:47:20 and 07:02:54 ± 00:53:05, respectively. The analysis revealed that participants had less awakenings during the CP trial (P < 0.05).

Subjective SOL did not differ between CP (19.9 ± 6.6 min) and CON (19.5 ± 14.6 min) (P = 0.972; r_rb = 0.010; 95% CI -4.64 to 6.14). Frequency of awakenings was significantly lower in CP (1.3 ± 1.5 counts) vs. CON (1.9 ± 0.6 counts) (P = 0.023; r_rb = 0.744; 95% CI -0.85 to -0.07) but wake length did not differ (CON = 10.3 ± 7.7 vs. CP = 9.6 ± 7.6) (P = 0.694; r_rb = 0.102; 95% CI -2.14 to 3.86).

Sleep variables derived from actigraphs are presented in Table 3. There were no significant differences for any variable (P > 0.05).

Table 4 shows the results from subjective surveys of sleepiness (KSS), fatigue, and sleep. There was a time effect for the KSS (P < 0.001) but there were no significant differences between supplements at any time-point (condition effect: P = 0.303; condition*time interaction effect; P = 0.217). Fatigue was higher mid-day than in the morning (P < 0.001) but there were no between trial differences (condition effect: P = 0.709; condition*time interaction effect; P = 0.275). There were no differences in subjective sleep quality between the CON and CP trials (P = 0.862; g = 0.048; 95 CI -0.28 to 0.24).

Table 5 shows the results from the cognitive function tests. There were time effects for average and fast, choice reaction time, which decreased Post (P < 0.05) but no condition or interaction effects (P > 0.05). There was a time*condition interaction effect for correct responses on the baseline Stroop test (P = 0.007) with post-hoc tests revealing that participants responses were more accurate in CP vs. CON (P = 0.009; g = 0.573; 95% CI 0.01 to 0.04).

Daily training load during the CON and CP trials was 419 ± 214 and 388 ± 269 AU, respectively; these were not significantly different (P = 0.578; g = 0.154; 95% CI -85.62 to 146.55). There were also no significant differences in actigraphy derived moderate to vigorous activity during the CON (1210 ± 395 min) and CP trials (1228 ± 416 min) (P = 0.653; g = 0.124; 95% CI -103.18 to 67.18).

Core temperature recordings were missing at random for 7/117 time-points in CON and CP. As shown in Fig. 1, there was a time effect (P < 0.001) whereby core temperature decreased over-time, but there was no condition (P = 0.409) or condition*time interaction effects (P = 0.728).

Results from the blood and urine analysis are displayed in Table 6. Due to issues with analysis, 7/52 samples were not analysed for normetanephrine. There was a time effect for cortisol (P < 0.001), which increased Post in both conditions (P < 0.001) but there were no significant condition or interaction effects. There were no time, condition or interaction effects for IL-6, hs-CRP or normetanephrine:creatinine ratio (P > 0.05).